Peptides for discrimination of prions

ABSTRACT

The aim of the present invention is to provide a non-intrusive way to isolate, concentrate and monitor the TSE disease-related pathogenic prion protein. The invention described several peptides and their ability to capture PrP Sc  from brain homogenate of prion disease infected animal and human. These eight peptides do not capture cellular prion protein from individual with no prion disease.

BACKGROUND

Prion diseases or transmissible spongiform encephalopathies (TSE) havecaptured the interest of the scientific community, not only because theychallenged conventional thought about the nature of infectious agents,but also because of the potentially serious public health threat theypose to food and blood safety. Furthermore, the possibility of diseasetransmission between animals and humans has been dramaticallyunderscored in recent years by the epidemic occurrence of a new variantdisease form, vCJD. The present invention described the use of syntheticpeptides for the diagnosis of Transmissible Spongiform Encephalopathies(TSE) diseases in animals and humans.

Prions—A Pathogenic Agent Causing TSE

Transmissible spongiform encephalopathies (TSEs) comprise a group ofrapidly progressing, neurodegenerative fatal diseases that affect bothhumans and animals. TSEs have clinical and neuropathologicalcharacteristics which include devastating dementia, pyramidal andextrapyramidal signs with myoclonus, multifocal spongiform changes,astrogliosis, amyloid plaques, neuronal loss, absence of inflammatoryreaction and are usually characterized by a long incubation period.

It was once suggested that TSE diseases might be caused by “slowviruses” or viroids (Gaidusek 1977). However, the extreme resistance ofscrapie infectivity to radiation, nucleases, and other reagents damagingto genetic materials are inconsistent with the “virus” theory. All these“unusual” characteristics of the TSE infectious agent led Dr. StanleyPrusiner to propose the concept of “prions” in 1982 (Prusiner 1982).Prion (PrP), which stands for nucleic acid-free proteinaceous infectiousparticle, is a glycoprotein present in. humans and animals. The cellularform of this protein (PrP^(c)) has two N-link glycosylation sites and aGPI anchor at the C-terminus. It has been most commonly found inneurons, and, to a much lower extent, it has also been found in othercells such as leucocytes, monocytes and platelets (Holada 2000). Thetransmissible scrapie disease form of the prion protein (PrP^(Sc)) is aprotease resistant isoform of its cellular precursor and ispredominantly found in brain. At much lower level, it has also beenfound in tonsil, spleen, and lymph nodes in vCJD patients (Parizek2001). As a result of Prusiner's concept of the “prion” as an infectiousagent responsible for scrapie disease, and by extension, that of all TSEdiseases gave rise to the notion of what are commonly referred to asPrion diseases to describe a class of pathologies believed to be linkedto this protein.

Human prion disease is known as Creutzfeldt-Jakob disease (CJD) andusually affects elderly persons. Depending on disease phenotype,sporadic (sCJD) accounts for most of CJD, while about 10% are familialCJD (fCJD). Only recently, a new variant of CJD (vCJD) has emerged whichoccurs in young adults. Since 1995, more than 100 cases have beenreported. Prion diseases affect many species of domestic and wildanimals, manifestations of which are often given unique names, forexample, scrapie in sheep and goats (McGowan 1922), chronic wastingdisease (CWD, Williams 1980) in cervids and bovine spongiformencephalopathy (BSE, or “mad cow” disease, Wells 1987) in cattle.

Characteristics of PrP^(C) and PrP^(Sc)

According to the Prusiner's “protein only” hypothesis, the transmissiblepathogen causing prion diseases is a protein. The major component of theprion appears to be abnormally folded PrP, designated as PrP^(Sc)(disease-specific, “scrapie” isoform). It is believed that PrP^(Sc) isgenerated from its normal counterpart, PrPc, through a change inconformation (Cohen, 1998). Although there is still ambiguity concerningthe mechanism of the conversion, it is still broadly accepted that inthe presence of PrP^(Sc), normal PrP^(C), acting as a substrate,undergoes a conformational structure change, and becomes PrP^(Sc)through an autocatalytic process and results in PrP^(Sc) aggregation andamyloid rod formation, hence causing cell death (Hope 1986, Horwich1997). The structural change from PrP^(C) to PrP^(Sc) is most supportedby a crucial conformational change, involving a substantial increase inthe amount of Beta-sheet structure of the protein, with possibly a smalldecrease in the amount of alpha-helix, indicated by circular dichroismand infrared spectroscopy (Pan 1993, Caughey 1991). It appears that theefficiency of conversion from PrP^(C) into PrP^(Sc) depends on thedegree of sequence homology between the two PrP conformers. Because thisunique mechanism, prion diseases can be readily transmitted within thesame species, and when the PrP sequence homology is sufficient, fromspecies to species (Raymond, 2000). Therefore, if correct, the proposedmechanism of transmission of prion diseases provides the potential totrigger epidemics by transmission of these brain disorders betweenanimals and humans.

Protease resistance is another characteristic that distinguishesPrP^(Sc) from PrP^(C). In cultured cells and brain or in samples frommany patients with GSS, PrPSc is smaller than its cellular precursorPrP^(C). Even though cellular prion and scrapie prion are two isoform ofsame PRNP genomic product, PrP^(C) is completely degraded by ProteinaseK treatment while PrP^(Sc) undergoes only limited digestion. Thedigestion yields a form of protein referred to as PrP 27-30 in which theN-terminus has been removed. PrP 27-30 has been postulated to be thePrP^(Sc) core required for PrP^(C) hosted PrP^(Sc) replication. Theprotease treated prion molecule, PrP 27-30 or PrP^(res), is tightlylinked to scrapie infectivity (Gabizon 1988), and provides additionalevidence that PrP^(Sc) is an infectious protein.

An additional attribute, perhaps linked to the significant increase inBeta-sheet structure and concomitant protease-resistance, is theobserved difference in solubility between PrP^(Sc) and PrP^(C). WhilePrP^(C) is a soluble protein, the PrP^(Sc) isoform is highly insoluble.Furthermore, PrP^(C) is found attached to the surface of neurons througha GPI tail anchored into membrane (Shyng 1994) while PrP^(res) is foundin the cytoplasm of affected cells (Taraboulos 1990), most likelyassociated with late endosomal and lysosomal compartments (Arnold 1995),and PrP^(Sc) is also localized in amorphous aggregates in enrichedfractions from infected brain (Meyer 1986).

There is mounting evidence indicating a tight linkage between scrapieinfectivity and PrP 27-30. Even in the purest samples, the estimatedratio of PrP molecules to infectious units is ˜10⁴ to 10⁵ (Horwich 1997,Bolton 2001). At such low levels of infectivity, it is possible thatother components, co-factors, or covalent modifications, are requiredfor infectivity. The transgenic studies on the susceptibility of miceexpressing chimeric human-mouse PrP^(C) suggest the presence of at leastone host factor other than PrP^(C), tentatively termed factor X, whichmight function as a molecular chaperone in the formation of PrP^(Sc)(Telling 1995).

Infectivity and Transmissibility of Prion Diseases

Prion diseases are transmissible. The transmissibility of animal priondiseases has long been established experimentally by inoculation ofbrain homogenates from affected animals into healthy ones, such scrapiefrom sheep to sheep (Cuillé 1936) and across species to goat (Paftison1957), kuru and CJD from humans to chimpanzees (Gajdusek 1966, Gibbs1968), The significant breakthrough was the successful transmission ofscrapie to mice (Chandler 1961) which greatly facilitated TSE researchby providing an experimental model. The cause of recent BSE in cattleand new variant CJD in human (vCJD) was considered a consequence ofdietary exposure to the mix of scrapie sheep carcasses rendered foranimal feed in the case of BSE (Brown 1997) and to beef from cattleaffected with BSE in the case of vCJD (Bruce 1997). The link betweenvCJD and BSE is further supported by the neuropathologic evidenceobtained from BSE-adapted macaques, the nearest model to humans, andfrom the study on inbred mice inoculated with the agent causing BSE andVCJD (Lasmézas 1996). Of particular concern to an epidemic expanding ofCWD in mule deer and elk in North America is whether CWD, like BSE,could be transmitted to humans who may be exposed to the disease throughhunting, or handling and eating infected meat. Tragic, unintendedtransmission of prion disease in humans has been documented, such as thekuru epidemic caused by cannibalistic ingestion of brain tissue from thedeceased, and the iatrogenic transmission of CJD through the use ofhormones, tissue transplants, and contaminated medical devices.

There is no hard evidence indicating any of CJD diseases is related toanimal TSEs that may have crossed species barriers. The epidemic of kuruhas provided the largest body of evidence of acquired human priondisease. Although no vCJD patient has been documented as a victim ofhuman-to-human transmission, the close link between BSE and vCJDattracted considerable attention. Concerns about human infection havebeen based on the observation that PrP^(Sc) is readily detectable in BSEand vCJD lymphoreticular tissues but not in classic CJD (Hill 1997),followed by the presumption that scrapie pathogen from sheep passage tocattle may have altered host range and become more adaptable to human.Experimental precedents for such behavior are well known: passage ofmouse-adapted strains of scrapie through hamsters altered theirtransmissibility on back passage to mice (Kimberlin 1987, Kimberlin1989); human strains of kuru or CJD did not transmit to ferrets or goatsuntil passaged through primates or cats (Gibbs 1979); and a bovinestrain of BSE did not transmit to hamsters until passaged through mice(Foster 1994). Alternatively, if BSE originated from a spontaneousmutation in cattle, experimental studies of species susceptibility tothis new strain of transmissible spongiform encephalopathy (TSE) had notsufficiently advanced to predict that humans would not be susceptible.

Study on human CJD and vCJD disease indicated that genomicsusceptibility might yet be another factor that may influence the spreadof TSE in humans. The majority of sporadic CJD patients were found to behomozygous for Met/Met or for Val/Val at codon 129 (Belay 1999).Nevertheless, all reported vCJD cases have been found to be homozygousfor Met/Met.

The size and duration of vCJD epidemic still remains uncertain.Depending on the assumptions made and the modeling calculationsemployed, different predictions were proposed. One estimation of totalvCJD predicts as few as 205 cases (Valleron 2001). On the other hand,another prediction for vCJD mortality for the next 80 years ranges from50 to 50,000 if infection comes only from BSE. It could reach up to150,000 if BSE is proven to infect sheep and if subsequently it isallowed to enter human food chain (Ferguson 2002). Although it isimpossible to make accurate predictions if the necessary parameters areeither mistaken or not available, one thing is certain that if vCJDinfectivity is present in blood, any prediction will be anunderestimate. In addition, vCJD has been proven to be a new diseaseentity and not simply the result of increased surveillance of CJD inhumans (Hillier 2002).

Countermeasures have been taken by government to eliminate the spread ofBSE incidence. Ruminant protein feed was banned in US and UK (1988). Aseries of measures have also been taken to prevent potentially infectedmeat from entering human food chain. To further reduce the human risk,FDA and CBER has issued a new policy in August 2001, which indefinitelydefers any donor who stayed cumulative ≧6 month during 1980-1996 in theUnited Kingdom (FDA 2001).

Diagnostic Assay for Prion Disease

The development of sensitive and reliable assays for prion detection isabsolutely essential for disease surveillance, risk assessment, and whencombined with future therapeutics, for disease prevention anderadication. Currently, there are basically three assay formats fordiagnosis of prion diseases. (1) Animal infectivity bioassays are by farthe most sensitive method for the measurement of infectious prion inexperimental scrapie in rodents, usually accomplished by intracerebralinjection of brain homogenates from sick animals into recipient animals.However, the quantitative measurement of prion disease infectivity indifferent animal species is limited due to the “species barrier” anddistinct prion “strains” exist that differ in terms of pathology,incubation time, and molecular characteristics of PrP^(Sc) . Therefore,this time-consuming, expensive postmortem diagnosis is mostly used as aresearch tool for distinguishing different prion strains in rodents, andserves as a reference for calibration of infectious brain material. (2)Current PrP Immunoassays are based on the detection ofprotease-resistant PrP^(Sc), the only known molecular hallmark of allprion diseases. For many years, the detection of PrP^(Sc) byimmunochemical methods (immunohistochemistry and Western blotting) hasprovided the most accurate diagnosis for prion diseases in animals andhumans (Schaller 1999, Biffiger 2002). They are widely used forpostmortem diagnosis. Many monoclonal and polyclonal antibodies havebeen raised against various regions of PrP for this purpose such aswidely used 3F4, 6H4 described in U.S. Pat. No. 4,806,627 and EP0861900.However, only few were claimed to be able to discriminate betweenPrP^(C) (often present in much larger quantities) and PrP^(Sc).Monoclonal antibodies reported by Korth in 1997 and by Paramithiotis in2003 were both IgMs and no diagnosis assay was developed by theseantibodies. Consequently, almost all current immunochemical methodsrequire a step to reduce or eliminate a PrP^(C), usually by nonspecificproteolysis such as proteinase K (PK) digestion, prior to the detectionof PrP^(Sc) . Such pretreatment cleaves the first 60-70 residues fromPrP^(Sc) to yield a PK-resistant PrP27kDa-30 kDa core called PrP^(res).Anti-PrP antibodies that recognize the remaining C-terminal region ofthe protein can then be used to detect the N-terminally truncatedPrP^(Sc), or PrP^(res), present only in pathological samples. Forimmunohistochemical staining, tissue sections are also pretreated,usually by acid hydrolysis to reduce the PrP^(C) related background.Therefore, PrP^(res) is a surrogate for the precursor PrP^(Sc) in theseimmunoassays. Among the various immunoassay formats, Western blottinghas the advantage of revealing detailed molecular patterns of PrP basedon the migration of di-, mono- and unglycosylated PrP bands. This methodalso has been widely used for distinguishing distinct brain PrP^(res)subtypes in human and animal prion diseases. Besides Western blot, otherassay formats have now been developed for higher sample throughput,increased sensitivity, and better quantification, including traditionalELISA, dissociation-enhanced lanthanide-fluorescence-immunoassay(DELFIA, Barnard 2000 and a method described in US20020137114A1) andconformation-dependent immunoassay (CDI) combined with ELISA andfluorescence detection (Safar 1998, US 20010001061A1, US20020001817A1).However, regardless of the format, PrP^(Sc) can be differentiated fromPr^(PC) only after the mandatory PK digestion, which may be difficult tooptimize for disparate biological samples. (3) Other methods have alsobeen described for sample treatment including immunohistochemistry ofthird eyelid lymphoid tissue for preclinical diagnosis of ovine scrapie(O'Rourke 2000, U.S. Pat. No. 6,165,784, U.S. Pat. No. 6,261,790),chemical treatment with sodium phosphotungstate to enrich PrP^(Sc) frombrain and from other peripheral tissue homogenates (Wadsworth 2001), anddetection of a new isoform of the prion protein in the urine of infectedanimals and humans (Shaked 2001b, WO0233420A2). Other detection systemswere also documented including capillary electrophoresis, and Fouriertransform infrared spectroscopy. These methods are still in theirinitial stages of development and are technically complex. In additionto the traditional identification of pathogenic prion by eliminatingcellular prion followed by non-discriminatory anti-prion antibodyrecognition, other reagents were found to be able to differentiatePrP^(Sc) from PrP^(C), such as plasminogen and fibrinogen. The evidenceprovided suggested that a property common to PrP^(Sc) of variousspecies, rather than prion primary sequence or the specific tertiarystructure of individual PrP^(Sc) molecules, could be responsible forbinding to plasminogen (Fischer 2000, Maissen 2001). The application forthe use of plasminogen and other serum/plasma proteins for the captureand detection of pathogenic prion protein has been described inWO0200713 and in US20010053533A1 (Aguzzi 2001).

All current manufactured prion diagnosis assays use brain tissue as asample source. The European Commission in 1999 evaluated 4 BSE test kitsfrom different manufacturers (Moynagh 1999). They all required aseparate sample preparation procedure. Depending on the kitinstructions, the brain tissue homogenate needed to be processed,including denaturation, PK digestion or PrP^(Sc) enrichment. The assaydetection systems employed in DELFA, immunoblot, or in plate ELISAformats used either chemiluminescent or a colorimetric substrate.

Challenges to Antemortem Diagnosis of Prion Disease

An issue common to PrP^(Sc)-based antemortem assays is whether PrP^(Sc)is present in peripheral tissues or body fluids. Because of technicaldifficulties, little experimental data on the presence of PrP^(Sc) orits associated infectivity in body fluids are available, and thissubject remains controversial. In the hamster model of scrapie, a lowlevel of infectivity can be detected in blood. Although the infectivityin lymphocyte-rich buffy-coat derived from diseased hamster blood isgreater than in plasma, it only accounts for relatively a small portionwhen compared to whole blood inoculums. The molecular definition of thisinfectious agent present in the blood is not clear. Searching for riskfactors and possible sources of infection in sporadic CJD patientsrevealed no significant correlation of disease to diet, bloodtransfusion or receiving other blood product. Although early reportsindicated the possible presence of infectivity in blood obtained fromCJD patients after intracerebral inoculation to mice (Manuelidis 1985,Tateishi 1985), The highest amount of infectivity or PrP^(res) isinvariably found in the central nervous system (CNS), but notconsistently found in peripheral tissues in classical human priondiseases, except in the case of vCJD. The presence of readily detectablePrP^(Sc) in the peripheral lymphoreticular tissues such as tonsils,spleen, and lymph nodes of vCJD patients has raised a serious concernthat abundant amounts of PrP^(res) present in lymphoreticular tissuescould interact with the circulatory system, and as a consequence, traceamounts of PrP^(Sc) may be present in blood of vCJD patients forpossible blood transmission. Other TSE infectivity in blood has alsobeen demonstrated in various experimental animals. Most blood forinfectivity studies was obtained from TSE -adapted rodents such as miceand hamsters. Mice-adapted BSE, mice-adapted vCJD has been establishedthrough intracerebral and intravenous transmission. The only exceptionmodel was a study conducted in the sheep. In this experiment, a sheeptransfused with whole blood, taken from another sheep inoculated withBSE brain lysate, developed symptoms of BSE (Houston 2000, Hunter 2002).However, these experimental results yet need to be fully evaluated. Itis anticipated that finding of such infectious agent in blood would helpus to better understand the relationship between PrP^(Sc) and TSEdisease.

It is important to note that the harsh sample treatment to eliminatePrP^(C) background may not be suitable for other peripheral tissuespecimens or body fluids due to differing protein content, thedifficulty of applying this assay to large numbers of samples, theinevitable elimination of protease-sensitive PrP^(Sc) foldingintermediates or even a fraction of authentic PrP^(Sc), thus reducingthe sensitivity of detection. This may be especially relevant for assaysusing peripheral tissues and body fluids, as only low levels of PrP^(Sc)may be present (Horiuchi 1999, Jackson1999, Swietnicki 2000). Theseconcerns necessitate the development of immunological reagents with ahigh affinity for PrP^(Sc), allowing for specific detection without theneed for proteolytic treatment.

Discovery of Novel Capture Reagent for PrP^(Sc) Detection

Whether or not one accepts the “protein only” or “prion only”hypothesis, there are continuous efforts underway to search for agentsor molecules other than prion that may contribute to the pathogenesis ofprion disease. This search is driven by many unanswered questions. Forexample, synthesized prion protein, free of any contamination, does notcause disease; the mechanism that triggers the conversion of normalPrP^(C) to the pathogenic PrP^(Sc) isoform is unknown. Anotherunresolved question involves the various prion disease phenotypesobserved in animals and humans, defined by disease incubation period,glycosylation level and lesion patterns. After serial passage in inbredmice homozygous for a single PRNP genotype, all the scrapie strainsretained their original disease profile. These observations ledinvestigators to question whether varied phenotypic strains weredominated by different conformational isoforms of same cellular prionprecursor, or whether there is another factor that determines thephenotype of the inheritable strain. In fact, in vitro conversion modelsPrP^(res) formed in cell-free reactions has never been shown toconstitute new TSE infectivity in animals (Caughey 2003). Thesequestions led many to believe that there is a missing element, dubbed“protein X” as Prusiner suggested, yet to be discovered.

The presence of a tightly bound RNA or DNA molecule in the prionparticle was proposed to explain propagation of different strains ofscrapie agent with distinct phenotypes in animals homozygous for thePRNP gene (Weissmann 1991). Analysis of highly purified scrapie prionsby return refocusing gel electrophoresis revealed the small size ofremaining nucleic acids (Kellings 1992). In a recent report, however,Narang indicated that animals inoculated with ssDNA purified fromscrapie-hamster brains mixed with non-pathogenic prion developedclinical disease (Narang 2002). Based on his findings, he postulatedthat the “accessory protein” coded by the ssDNA may be involved inPrP^(C) to PrP^(Sc) conversion. Based on those in vitro conformation andconversion studies, it was hypothesized that DNA would act as a guardianof the PrP^(Sc) conformation as well as a catalyst to facilitatePrP^(Sc) conversion and aggregation (Cordeiro 2001). Most recently, itwas reported that stoichiometric transformation of PrP^(C) to PrP^(res)in vitro requires specific RNA molecules (Deleault 2003). Theanti-nucleic acid monoclonal antibody developed by Ortho-ClinicalDiagnostics that can discriminatively capture PrP^(Sc) but not PrP^(C)(U.S. 60/434,627, U.S. 60/446,217) is another evidence to demonstratethe association of PrP^(Sc) to nucleic acids.

It is known that PrP^(Sc) isolated from diseased brain is alsoassociated with a variety of glycans. Those include 1,4-linked glucoseunits in prion rods, sphingolipids, polysaccharides and other membranecomponents in PrP^(Sc) aggregates (Appel 1999, Klein 1998), and sulfatedproteoglygan in prion amyloid plaques (Snow 1990), a property that hasbeen exploited in immunohistochemistry, where binding by heparan sulfateantibodies (anti-HS) and heparan sulfate proteoglycan antibodies(anti-HSPG), has been shown to correlate with abnormal PrP as early as70 days post-infection and throughout the course of the disease (McBride1998). Through a mechanism that is perhaps different from that by whichnucleic acids participate in the conversion of PrP^(C) to PrP^(Sc),glycan also convert cellular prion protein into Beta-sheet conformation.In vitro conversion from PrP^(C) to PrP^(Sc) and in prion infectivityreconstitution experiments, sulfate glycans have been shown either tofacilitate the conversion or to escalate infectivity (Wong 2001, Shaked2001a, Diaz-Nido 2002). With recombinant GST::full-lengtht prion andGST::prion fragment, Warner recently demonstrated direct binding ofrecombinant prion to heparin and heparan sulfate (Warner 2002). Thepeptide region 23-52 in prion sequence was positive in all HS and HSPGbinding tests. Since the peptide failed to compete with full-lengthprion for binding to heparin, the author suggested that there might beanother major GAG-binding site in intact PrP^(C). Another interestingobservation is that plasminogen has been reported to bind brain-derivedPrP^(Sc), but not to PrP^(C). Although it has not been demonstrated thatplasminogen has a direct interaction with PrP^(Sc), a binding site issuggested within the Kringle region of plasminogen, a region that has aknown affinity for heparin. Another noteworthy observation is that GAGsfrom different species (bovine and porcine) or from different organs(lung, kidney and intestine) have shown different affinities for prionbinding. The difference in affinity may be due to prion sequence itself,or may depend on the presence of particular sugar unit in the testedGAGs.

In light of these observations, a number of peptides were proposed thatare designed to selectively capture PrP^(Sc) but not PrP^(C) throughpeptide affinity to unique PrP^(Sc) conformation or to PrP^(Sc)associated molecules. They were screened by the ability to identify andto capture PrP^(Sc) from homogenates of diseased brains byimmunoprecipitation without protease pretreatment.

(1) Heparin/Heparin Sulfate Binding Domain Peptides:

Glycans (GAG) such as heparin and heparin sulfate were associated withPrP^(Sc) amyloid aggregates. Because the association affinity was muchhigher in GAG::PrP^(Sc) than in GAG::PrP^(C) it is possible to usepeptides characterized as glycan binding domain to selectively capturePrP^(Sc) but not PrP^(C) . For this reason, peptides described asheparin or heparin sulfate binding domain were synthesized: (1) WQPPRARIof carboxy-terminal fibronectin (Woods 1993, Hines 1994) (2)NWCKRGRKNCKTH of amyloid protein precursor (Small 1994), (3) NYKKPKLG ofN-terminal fibroblast growth factor (FGF)-1 (Lou 1996) and (4)KDFLSIELVRGRVK of the C-terminal G-domain of the laminin alphal chain(Yoshida 1999).

(2) “Condensed” Kringle Peptides:

Kringle region was involved in the selective binding of plasminogen toPrP^(Sc). It was also known that Kringle region had heparin/heparinsulfate binding activity (Mizuno 1994) for which positively chargedamino acids (such as Arg and Lys) were involved (Soeda 1989). In aseparate publication, two conserved tripeptides “YYR”, or rather threediscontinued “YYX” in prion sequence as suggested, were found tointeract with PrP^(Sc) but not interact with PrP^(C) (Paramithiotis2003, WO0078344A1). Interestingly found in human plasminogen sequence,there were four Tyr-Arg-Gly sequences in four out of five Kringleregions (Kringle region 1, 3, 4 and 5); there were six Tyr-Arg orArg-Tyr or Arg-Lys sequences in plasminogen. Furthermore, severaldistant Tyr, Arg or Lys residues were brought closely together bydisulphide bridges formed in Kringle loops (FIG. 1). Based on theseobservations, it was postulated that amino acids Tyr, Arg and Lys inplasminogen Kringle region and in prion sequence, possibly through aninteraction to glycan that was associated with PrP^(Sc) complex, couldbe accounted for the selective binding to PrP^(Sc) . For this reason,two “condensed Kringle” peptides YRGYRGYRGYRG and YRGRYGYKGKYGYRG weresynthesized.

(3) Nucleic Acid Binding Peptides:

Nucleic acids are another category of molecules that associated withPrP^(Sc) aggregates. Anti-DNA antibodies have been used effectively tocapture PrP^(Sc). Histones are a group of proteins known to bind nuclearDNA. Therefore, three peptides (1) AQKKDGKKRKRSRKESYSIYV of H2B(21-41),(2) ARTKQTARKSTGGKAPRKQLA of H3(1-21), and (3) SGRGKGGKGLGKGGAKRHRKVLRof H4(2-24) were synthesized to evaluate their ability of the capture ofPrP^(Sc).

SUMMARY OF THE INVENTION

The aim of the present invention is to provide a non-intrusive way toisolate, concentrate and monitor the TSE disease-related pathogenicprion protein. The invention describes several peptides and theirability to capture PrP^(Sc) from brain homogenate of prion diseaseinfected animal and human. These eight peptides do not capture cellularprion protein from individual with no prion disease. The evidenceprovided in support of this invention demonstrated that PrP^(Sc) isassociated with high affinity to many other molecules such as nucleicacid and glycans as investigated. The evidence also demonstrated thatthe association was strong, resistant to PK digestion treatment, andthat PrP^(Sc) could be readily isolated by peptides through recognitionof such associated binding partner.

This invention relates to the use of described peptides to capturePrP^(Sc) through glycans or nucleic acids associated with high affinityto PrP^(Sc), in combination with any prion sequence-specific antibodyfor the detection of PrP^(Sc).

In another aspect, this invention relates to the peptides, as describedabove, that preferably binds to pathogenic prion protein but not to thenormal cellular form of prion protein.

In another aspect, this invention relates to the peptides, as describedabove, for the detection of PrP^(Sc) through high affinity recognitionof associated glycans or nucleic acids in combination of prion sequencespecific antibodies.

In another aspect, this invention relates to the peptides, as describedabove, for the isolation, purification, capture, elimination andmonitoring PrP^(Sc) in biological reagent production.

In another aspect, this invention relates to compositions and kits fordetermining the presence of PrP^(Sc), comprising peptides, as describedabove, for either capture or for detection step in the assay procedure.

In another aspect, this invention relates to compositions and kits fordetermining the presence of PrP^(Sc) antibody produced in response tohigh affinity associated glycans or nucleic acids as a binding partnerto pathogenic prion protein.

In yet another aspect, this invention relates to anti-PrP^(Sc)antibodies and their production using the said glycans or nucleic acidsthat can interact with and/or associate with PrP^(Sc), and their use indetecting glycan::PrP^(Sc) or nucleic acid::PrP^(Sc) complex and priondisease infection.

In another aspect, this invention relates to a non-harsh sampletreatment procedure involving glycanase or nuclease digestion for thebenefit of the use of described peptides as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of human plasminogen sequence and Kringleregions.

FIG. 2 is an illustration of an IP capture and immunoblot of hamsterPrP^(Sc).

FIG. 3 is an illustration of an IP capture and immunoblot of BSEPRP^(Sc).

FIG. 4 is an illustration of immunocapture of PRP^(Sc) from brains ofvCJD by peptides.

BRAIN HOMOGENATE PREPARATION

Normal and scrapie hamster brain lysate were obtained from BaltimoreResearch and Education Foundation as 10% whole-brain tissue homogenatein PBS (w/v). The lysate was further treated by adding 1/10 volume of10× detergent homogenate buffer, composed of 5% sodium deoxycolate and5% Igpal CA-630 (equivalent to NP-40) in PBS, incubating at 4C for 1hr., followed by centrifugation at 1000 g for 10 minutes. The resultingsupernatant was collected and used in the assay.

Normal and BSE bovine brain tissue were provided by VeterinaryLaboratories Agency (VLA), UK. Human vCJD and Lewy body dementia braintissue were provided by National CJD Surveillance Unit (NCJDSU), UK.Brain tissue was processed the same way (or similar) as hamster brainhomogenate preparation.

Synthetic Peptides:

Nine peptides were either synthesized by ResGen (currently a division ofInvitrogen Corporation), or purchased from Upstate Group, Inc. Eachpeptide was labeled with biotin either on the C-terminal Lysine bearingan aminohexanoyl spacer (K(Lc)), or at the N-terminal through anaminohexanoyl spacer (AMCAP). Sequence Peptide ID Peptide SequenceOrigin Made by SEQ ID No.: 1: WQPPRARIG-K(Lc)- Fibro- ResGen Biotinnectin SEQ ID No.: 2 Biotin-AMCAP- Amyloid ResGen NWCKRGRKNCKTH proteinprecursor SEQ ID No.: 3 Biotin-AMCAP- Fibro- ResGen NYKKPKLG blastgrowth factor (FGF)-1 SEQ ID No.: 4 Biotin-AMCAP- Laminin A ResGenKDFLSIELVRGRVK chain SEQ ID No.: 5 YRGYRGYRGYRG-K “con- ResGen(Lc)-Biotin densed” Kringle-A SEQ ID No.: 6 YRGRYGYKGKYGYRG- “con-ResGen K(Lc)-Biotin densed” Kringle-B SEQ ID No.: 7 AQKKDGKKRKRSRKESHuman Upstate YSIYV-GGK(Lc)- histon 2B Biotin SEQ ID No.: 8ARTKQTARKSTGGKAP Human Upstate RKQLA-GGK(Lc)- histon 3 Biotin SEQ IDNo.: 9 SGRGKGGKGLGKGGAK Human Upstate RHRKVLR-GSGSK histon 4 (Lc)-BiotinSEQ ID No.: 10: Biotin-AMCAP- VP16 ResGen TIADRYYRETAR protein, HSV-2Biotinylated peptide was dissolved in PBS at 1 mg/mL and stored at −20°C. until use.Coniugation of Biotinylated Peptide to Streptavidin Magnetic Beads: 0.5mL Dynabeads® M-280 Streptavidin (Dynal Biotech, NY, USA, Cat. # 112.06)were washed twice with PBS and the beads isolated from buffer with themagnet (Dynal Magnetic Particle Concentrator, MPC). 100 ug peptide and 1mL PBS was added to the washed beads. Incubation with rotation wasperformed at 37C for 1-2 hours. The beads were isolated from the bufferwith the MPC, washed twice with 1 ml PBS (0.1% BSA), and rotated for 5minutes at room temperature while washing. The peptide -conjugated beadswere then blocked for 2 hours, 37° C. with 1 mM Biotin in 0.2 MTris-HCI, pH 8.0, containing 0.1% BSA. The beads were subsequentlywashed 2 times with 1 ml PBS (0.1% BSA) and once with 1 ml PBS (0.1%BSA, 1% Tween 20) incubating each time for 10 minutes at room temp. Thebeads were then washed once with 1 ml PBS (0.1%BSA) and then stored in 1ml PBS (0.05% sodium azide) at 4° C.Proteinase K Digestion:

Conditions for the PK digestion of brain lysate: Brain homogenate wassuspended in PBS buffer with or without non-ionic detergent. The totalhomogenate protein concentration was no more than 2.5 mg/mL. PK (RocheDiagnostics, IN, USA, Cat. # 1373196) was added to a final concentrationof 50 ug/mL. Incubation was at 37C for 0.5 to 1 hour. Digestion wasstopped by adding Pefabloc SC (Roche Diagnostics, IN, USA, Cat. #1585916) to a final concentration of 4 mM.

Immunoprecipitation (IP), Non-Reducing Electrophoresis and ImmunoblotDetection of PrP^(Sc) :

Peptide conjugated magnetic beads were used to capture PrP^(Sc) frombrain homogenate by immunoprecipitation. The IP procedure consists ofthe following protocol: mix 50 uL peptide conjugated beads with PKtreated or non-PK treated brain homogenate in a total of 1 mL IP buffer(3% Tween20 and 3% Igpal CA-630 in PBS) and incubate at 25C for 2.5hours with rotation→Separate beads using MPC device and wash beads 3times of 30 second vortexing with IP wash buffer (2% Tween20 and 2%Igpal CA-630 in PBS)→Elute captured PrP^(Sc) by heating beads withNuPAGE sample buffer for 10-15 minutes. The eluted sample from IPcapture were loaded onto a 4-12% NuPAGE® Bis-Tris Gel (Invitrogen, CA,USA, Cat. # NP0302) and subjected to non-reducing electrophoresis at200V for 45 minutes. The immunoblot procedure was performed as follows:transfer separated proteins in the gel to a 0.2 um PVDF membrane(Invitrogen, Cat # LC2002) at 30V for 60 minutes→Block the membrane withBlocker™ Casein in TBS (0.05% Tween20) (Pierce Chemical Corp., IL, USA,Cat. # 37532) either at 25C for 1 hour with shaking or at 4C overnight.→As primary antibody, use 3F4 (Signet, MA, USA, Cat. # 9620-02) at1:3000 dilution or 6H4 (Prionics AG, Switzerland, Cat. # 01-011) at1:5000 dilution to detect PrP^(Sc). Incubate the membrane with dilutedprimary antibody in 10% Blocker™ Casein in TBST buffer (25 mM Tri-Cl,0.2M NaCl, 0.2% Tween20, pH 8.0) at 25C for 1 hour with shaking. →Wash3× 5 minutes with TBST buffer with shaking. →Incubate membrane withhorseradish peroxidase conjugated goat polyclonal anti-mouse IgG (H+L)(Jackson ImmunoResearch Laboratories, PA, USA, Cat. # 115-035-003) at1:10,000 to 1:30,000 dilution in 50% Blocker™ Casein in TBST buffer at25C for 1 hour with shaking. →Wash 6× 5 minutes with TBST buffer withshaking. →Add ECL chemiluminescence substrate (Amersham Biosciences, NJ,USA, Cat. # RPN2109) or SuperSignal West Dura chemiluminescencesubstrate (Pierce) on membrane to develop for 5 minutes. →Take image byexposure either to Bio Max MR-2 film (Kodak, NY, USA) or to the ChemiDocGel Documentation System (Bio-Rad, CA, USA).

Advantages.

The present invention uses peptide to capture PrP^(Sc) by recognition ofhigh affinity associated molecules in PrP^(Sc) complex such as glycans,nucleic acid. Because the tight association of these molecules only toPrP^(Sc) but not to PrP^(C), the present invention provided anon-intrusive means for the detection of PrP^(Sc) while no PK digestionor other protein modification procedure required. It is anticipated thatthe mild conditions will preserve the original structure andconformation of the pathogenic prion protein, thereby offeringopportunity to determine the presence of true PrP^(Sc) while minimizingthe generation of PrP^(Sc) due to harsh sample treatment.

The use of synthetic peptides offer advantages in that they display thebinding specificity but can also be easily handled in direct coating toa solid phase as well as be conjugated to link to signal given reagentssuch as horseradish peroxidase (HRP), or to be adopted into otherdesired diagnosis assay format.

Literature Cited.

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1. A reagent for the detection of PrP^(Sc) comprising any peptideselected from the group consisting of: (SEQ ID No.: 1) WQPPRARI; (SEQ IDNo.: 2) NWCKRGRKQCKTH; (SEQ ID No.: 3) NYKKPKL; (SEQ ID No.: 5)YRGYRGYRGYRG; (SEQ ID No.: 6) YRGYRGYKGKYRG; (SEQ ID No.: 7)AQKKDGKKRKRSRKESYSIYV; (SEQ ID No.: 8) ARTKQTARKSTGGKSPRKQLA; (SEQ IDNo.: 9) SGRGKGGKGLGKGGAKRHRKVLR; and.
 2. A method for detection ofPrP^(Sc) comprising: first contacting a sample with peptide claimed inclaim 1, then detecting PrP^(Sc).
 3. An immunoassay for detectingPrP^(Sc) comprising: providing a solid support having bound theretopeptide from claim 1, contacting the solid support with a sample,washing the support to remove any unbound sample, contacting the solidsupport with a prion specific antibody, and carrying out a detectionstep to determine if PrP^(Sc) are bound to the solid support.
 4. A kitfor the detection of PrP^(Sc) comprising a solid support having boundthereto peptide from claim 1, and a labeled prion specific antibody.